Unravelling the Link between Oligonucleotide Structure and Diastereomer Separation in Hydrophilic Interaction
Honorine Lardeux1,2, Kathrin Stavenhagen3, Clément Paris3
1School of Pharmaceutical Sciences, University of Geneva, CMU─Rue Michel Servet 1, Geneva 4 1211, Switzerland.
Abstract:
Therapeutic oligonucleotides (ONs) commonly incorporate phosphorothioate (PS) modifications. These introduce chiral centers and generate ON diastereomers. The increasing number of ONs undergoing clinical trials and reaching the market has led to a growing interest to better characterize the ON diastereomer composition, especially for small interfering ribonucleic acids (siRNAs). In this study, and for the first time, we identify higher-order structures as the major cause of ON diastereomer separation in hydrophilic interaction chromatography (HILIC). We have used conformational predictions and melting profiles of several representative full-length ONs to first analyze ON folding and then run mass spectrometry and HILIC to underpin the link between their folding and diastereomer separation. On top, we show how one can either enhance or suppress diastereomer separation depending on chromatographic settings, such as column temperature, pore size, stationary phase, mobile-phase ionic strength, and organic modifier. This work will significantly facilitate future HILIC-based characterization of PS-containing ONs; e.g., enabling monitoring of batch-to-batch diastereomer distributions in full-length siRNAs, a complex task that is now for the first time shown as possible on this delicate class of therapeutic double-stranded ONs.
Insights
Higher-order structures, not previously recognized, are the primary drivers of therapeutic oligonucleotide (ON) diastereomer separation using hydrophilic interaction chromatography (HILIC). This study reveals how ON folding influences separation and how chromatographic conditions can be tuned to control it.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Pharmaceutical Sciences
Background:
- Therapeutic oligonucleotides (ONs) often contain phosphorothioate (PS) modifications, introducing chirality and resulting in diastereomers.
- Accurate characterization of ON diastereomer composition is crucial, particularly for small interfering ribonucleic acids (siRNAs) in clinical development.
Purpose of the Study:
- To identify the primary cause of ON diastereomer separation in hydrophilic interaction chromatography (HILIC).
- To establish the relationship between oligonucleotide folding and diastereomer separation.
- To demonstrate control over diastereomer separation by adjusting chromatographic parameters.
Main Methods:
- Conformational predictions and melting profiles to analyze oligonucleotide folding.
- Mass spectrometry and HILIC to correlate folding with diastereomer separation.
- Systematic variation of chromatographic settings (temperature, pore size, stationary phase, ionic strength, organic modifier).
Main Results:
- Higher-order structures (folding) were identified as the major cause of ON diastereomer separation in HILIC.
- A direct link between ON folding and observed diastereomer separation was established.
- Methods to enhance or suppress diastereomer separation by manipulating chromatographic conditions were demonstrated.
Conclusions:
- This work elucidates the critical role of oligonucleotide higher-order structures in HILIC diastereomer separation.
- The findings provide a foundation for improved HILIC-based characterization of PS-containing therapeutic ONs, including batch-to-batch monitoring of siRNA diastereomer distributions.
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